Lignocellulosic biomass that is a renewable resource as an alternative to fossil fuels including petroleum and coal is evaluated as a major means for transition to biobased economy through conversion to bioalcohol as transportation fuel and lignocellulosic sugar which is an industrial fermentable sugar. Some developed countries including USA have already started the industrial production of bioethanol using lignocellulosic biomass as a raw material, and a recent report says that Renmatix and Sweetwater in USA commence the industrial production of industrial fermentable sugars from lignocellulosic biomass in 2017. The lignocellulosic biomass resource used is refinery by-products of woods and corns, and cellulose, which is one of the structural components of biomass, is a direct raw material of bioalcohol or industrial fermentable sugar.
Additionally, algal biomass including green algae and diatoms gains attention as third generation biomass and has been studied and developed to achieve commercialization, and it contains not only carbohydrate such as starch and cellulose but also protein and oil in the body and is a promising raw material of biofuels including bioethanol, biodiesel and foods. In algal biomass, green algae and diatoms mainly containing starch or cellulose do not have lignin in the body unlike lignocellulosic biomass, and do not need high-temperature pretreatment that is generally applied to lignocellulosic biomass, and carbohydrate present in the body, for example, starch and cellulose, is easily converted to monosaccharides by acids or starch degrading enzymes and cellulase.
To convert cellulose in biomass to glucose, acids or saccharification enzymes are added to the pretreated product containing cellulose as one of the main components obtained by pretreatment of biomass, and saccharification is carried out at a specified temperature for a predetermined time. The resulting saccharification products contain saccharification residues, for example, lignin, remaining nonhydrolyzed in solid state in a sugar solution in which monosaccharides of glucose or wood sugar produced by hydrolysis of cellulose or hemicellulose are dissolved. It is well known that the saccharification residues containing lignin as a main component have hydrophobic surface, so that the reduction of enzyme activity is occurred by irreversible adsorption of enzyme on it during enzymatic saccharification, which causes long saccharification time and yield loss.
To solve this problem, U.S. Pat. No. 8,728,320B discloses the addition of exogenous protein to a reaction system, binding to the surface of lignin to reduce the adsorption inactivation of enzymes, and adsorption of water-soluble lignin to protein and removal. But this technology does not yet propose a technical solution for effective solid-liquid separation to prepare a high concentration sugar solution and does not consider a method for reusing a considerable amount of active enzymes left after enzymatic saccharification.
In the case of bioethanol production with biomass as a raw material, many nutrients for microorganism cultivation including a source of nitrogen, for example, ammonia, are added to the saccharification products, and after inoculation of microorganisms and cultivation for a predetermined time, monosaccharides, for example, glucose, are converted to ethanol. Subsequently, after evaporation of ethanol by heating a fermentation broth in whole or in part, ethanol can selectively be recovered by condensing ethanol vapor.
In contrast, in the case of fermentable sugar (biosugar) production for microorganism cultivation using biomass as a raw material, a sugar solution is recovered from the saccharification products, and a purification process for removing impurities other than sugar and a concentration process for preparing a higher concentration sugar solution are additionally performed. To recover a sugar solution, it is necessary to remove insoluble solid particles from the saccharification products, and it is general to carry out filtration or centrifugation. For example, US2015/0344921A1 discloses centrifugation or filtration of saccharification products as they are, to recycle enzymes after saccharification. In addition, US2015/0344921A1 also discloses recovering and reusing enzymes from the filtrate.
However, when a centrifuge, for example, a centrifugal decanter, is used to recover a sugar solution from saccharification products containing a large amount of sugars, thus having higher specific gravity than water and containing even fine particles, it is necessary to spin it (for example, 60 or more minutes at 1,776×g per min) for a long time even by high speed rotation, and thus the energy cost is never low so that it may not be practical. Even in the case of membrane filtration to prepare a clear sugar solution, filtration itself is not easy and fine particles still remain after filtration. So, additional treatment, for example, microfiltration, is inevitable because fine particles clog the separator or filter cloth holes and the pressure increases rapidly. Additionally, when performing a solid-liquid separation of a slurry containing fine particles by filtration, it is known that a mineral additive, for example, a filter aid, is generally used.                For this reason, the filtration or centrifugation is occasionally carried out after aggregation of fine particles with an addition of polymeric flocculants. In this instance, the polymeric flocculants may be synthetic ionic or non-ionic chemicals with molecular weight of a few hundred thousand or more. And, the larger the amount of particles to be aggregated, the larger the amount of polymeric flocculants that are necessary to be used. Accordingly, when saccharification products produced from lignocellulosic biomass as a raw material contain a high concentration of sugar and a few % of insoluble saccharification residues as well, an amount of polymeric flocculants needed increases greatly, and the preparation cost of the resulting sugar solution inevitably increases. Additionally, this does not rule out the possibility of contamination of the sugar solution by the remaining polymer compound and its consequential limited use.        
Meanwhile, in the case of saccharification of biomass using enzymes, after enzymatic saccharification, the saccharification products may be heated to denature the enzymes that are already included in the saccharification products so that the enzymes have the ability to aggregate by themselves, and solid-liquid separation may be carried out (US2015/0354017A1). However, the saccharification enzymes (cellulase complex formulation) that are never cheap still remain in the saccharification products produced by enzymatic saccharification of pretreated lignocellulosic biomass, and it is known that a considerable amount of these enzymes still maintain enzyme activity even after saccharification (Novozymes's Product Sheet, Special Food/2001-08524-03.pdf). Like US Patent Publication No. US2015/0344921A1, the technology for separation, recovery and recycling of enzymes by ultrafiltration in the process of producing fermentable sugar using lignocellulosic biomass as a raw material is already suggested by many people. Accordingly, because thermal denaturation of saccharification enzymes by heating the enzymatic saccharification products of biomass and the use of the aggregation ability of the denatured protein for solid-liquid separation results in the giving up of an opportunity to use a considerable amount of active enzymes left even after enzymatic saccharification, it is not desirable.
Accordingly, after conducting a lot of efforts to develop a technology that prepares a high concentration sugar solution by easily performing a solid-liquid separation of saccharification products of biomass without using the aggregation ability of denatured enzymes, and recovers a sugar solution containing enzymes by this effective solid-liquid separation to reuse a considerable amount of active enzymes left even after enzymatic saccharification, the inventors finally completed the present invention.